Surveillance of tick-borne bacteria infection in ticks and forestry populations in Inner Mongolia, China.

Ticks are one of the most important vectors that can transmit pathogens to animals and human beings. This study investigated the dominant tick-borne bacteria carried by ticks and tick-borne infections in forestry populations in Arxan, Inner Mongolia, China. Ticks were collected by flagging from May 2020 to May 2021, and blood samples were collected from individuals at high risk of acquiring tick-borne diseases from March 2022 to August 2023. The pooled DNA samples of ticks were analyzed to reveal the presence of tick-borne bacteria using high-throughput sequencing of the 16S rDNA V3-V4 region, and species-specific polymerase chain reaction (PCR) related to sequencing was performed to confirm the presence of pathogenic bacteria in individual ticks and human blood samples. All sera samples were examined for anti-SFGR using ELISA and anti-B. burgdorferi using IFA and WB. A total of 295 ticks (282 Ixodes persulcatus and 13 Dermacentor silvarum) and 245 human blood samples were collected. Rickettsia, Anaplasma, Borrelia miyamotoi, and Coxiella endosymbiont were identified in I. persulcatus by high-throughput sequencing, while Candidatus R. tarasevichiae (89.00%, 89/100), B. garinii (17.00%, 17/100), B. afzelii (7.00%, 7/100), and B. miyamotoi (7.00%, 7/100) were detected in I. persulcatus, as well the dual co-infection with Candidatus R. tarasevichiae and B. garinii were detected in 13.00% (13/100) of I. persulcatus. Of the 245 individuals, B. garinii (4.90%, 12/245), R. slovaca (0.82%, 2/245), and C. burnetii (0.41%, 1/245) were detected by PCR, and the sequences of the target genes of B. garinii detected in humans were identical to those detected in I. persulcatus. The seroprevalence of anti-SFGR and anti-B. burgdorferi was 5.71% and 13.47%, respectively. This study demonstrated that Candidatus R. tarasevichiae and B. garinii were the dominant tick-borne bacteria in I. persulcatus from Arxan, and that dual co-infection with Candidatus R. tarasevichiae and B. garinii was frequent. This is the first time that B. miyamotoi has been identified in ticks from Arxan and R. solvaca has been detected in humans from Inner Mongolia. More importantly, this study demonstrated the transmission of B. garinii from ticks to humans in Arxan, suggesting that long-term monitoring of tick-borne pathogens in ticks and humans is important for the prevention and control of tick-borne diseases.

Large-scale genomic survey and characterization of mcr genes carried by foodborne Cronobacter isolates.

IMPORTANCE: Cronobacter is an emerging foodborne opportunistic pathogen, which can cause neonatal meningitis, bacteremia, and NEC by contaminating food. However, the entire picture of foodborne Cronobacter carriage of the mcr genes is not known. Here, we investigated the mcr genes of Cronobacter isolates by whole-genome sequencing and found 133 previously undescribed Cronobacter isolates carrying mcr genes. Further genomic analysis revealed that these mcr genes mainly belonged to the mcr-9 and mcr-10. Genomic analysis of the flanking structures of mcr genes revealed that two core flanking structures were prevalent in foodborne Cronobacter isolates, and the flanking structure carrying IS1R was found for the first time in this study.

Molecular characteristics and antibiotic resistance mechanisms of clindamycin-resistant Streptococcus agalactiae isolates in China.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a major cause of neonatal infections with high morbidity and mortality, and clindamycin is the main antibiotic used to treat GBS infections in patients allergic to penicillin. We aimed to analyse the antibiotic sensitivity, sequence types, serotypes, virulence factors, and antibiotic resistance mechanisms of clinically isolated clindamycin-resistant S. agalactiae and provide basic data for the treatment, prevention, and control of clinical infection of S. agalactiae. A total of 110 strains of clindamycin-resistant S. agalactiae were collected from two tertiary hospitals in Hebei, China. We performed antibiotic sensitivity tests for 11 antibiotics on these strains and whole-genome sequencing analysis. All the strains were susceptible to penicillin, ampicillin, linezolid, vancomycin, tigecycline, and quinupristin-dalfopristin. Resistance to erythromycin, levofloxacin, tetracycline, and chloramphenicol were also observed. Genome sequence analysis revealed that all strains belonged to 12 sequence types (STs) related to six cloning complexes (CCs), namely CC10, CC19, CC23, CC651, CC1, and CC17. Five serotypes were identified, including IA, IB, II, III, and V. The most prominent resistance genes were mreA (100%) and ermB (81.8%). Furthermore, cfb, cylE, pavA and the gene cluster related to the pili were 100% present in all strains, followed by lmb (95.5%) and srr1 (67.2%). This study found that clindamycin-resistant S. agalactiae showed polymorphisms in molecular types and serotypes. Furthermore, multiple virulence factor genes have been identified in their genomes.

Investigation of bacterial and fungal population structure on environmental surfaces of three medical institutions during the COVID-19 pandemic.

Objectives: To evaluate the population structure of environmental bacteria and fungi in three different types of medical institutions and the potential risks due to antibiotic resistance during the coronavirus disease 2019 (COVID-19) pandemic. Methods: One hundred twenty-six environmental surface samples were collected from three medical institutions during the COVID-19 pandemic. A total of 6,093 and 13,514 representative sequences of 16S and ITS ribosomal RNA (rRNA) were obtained by amplicon sequencing analysis. The functional prediction was performed using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States tool based on the Greengenes database and the FAPROTAX database. Results: On environmental surfaces in three medical institutions during the COVID-19 pandemic, Firmicutes (51.6%) and Bacteroidetes (25%) were the dominant bacteria, while Ascomycota (39.4%) and Basidiomycota (14.2%) were the dominant fungi. A number of potential bacterial and fungal pathogens were successfully identified by the metagenomic approach. Furthermore, compared with the bacterial results, the fungi showed a generally closer Bray Curtis distance between samples. The overall ratio of Gram-negative bacteria to Gram-positive bacteria was about 3:7. The proportion of stress-tolerant bacteria in medical institutions A, B and C reached 88.9, 93.0 and 93.8%, respectively. Anaerobic bacteria accounted for 39.6% in outdoor environments, 77.7% in public areas, 87.9% in inpatient areas and 79.6% in restricted areas. Finally, the ß-Lactam resistance pathway and polymyxin resistance pathway were revealed through functional prediction. Conclusion: We described the microbial population structure changes in three different types of medical institutions using the metagenomic approach during the COVID-19 pandemic. We found that the disinfection measures performed by three healthcare facilities may be effective on the "ESKAPE" pathogens, but less effective on fungal pathogens. Moreover, emphasis should be given to the prevention and control of ß-lactam and polymyxin antibiotics resistance bacteria during the COVID-19 pandemic.

Epidemiological Investigation of Hospital Transmission of Corynebacterium striatum Infection by Core Genome Multilocus Sequence Typing Approach.

Corynebacterium striatum has recently received increasing attention due to its multiple antimicrobial resistances and its role as an invasive infection/outbreak agent. Recently, whole-genome sequencing (WGS)-based core genome multilocus sequence typing (cgMLST) has been used in epidemiological studies of specific human pathogens. However, this method has not been reported in studies of C. striatum. In this work, we aim to propose a cgMLST scheme for C. striatum. All publicly available C. striatum genomes, 30 C. striatum strains isolated from the same hospital, and 1 epidemiologically unrelated outgroup C. striatum strain were used to establish a cgMLST scheme targeting 1,795 genes (hereinafter referred to as 1,795-cgMLST). The genotyping results of cgMLST showed good congruence with core genome-based single-nucleotide polymorphism typing in terms of tree topology. In addition, the cgMLST provided a greater discrimination than the MLST method based on 6 housekeeping genes (gyrA, gyrB, hsp65, rpoB, secA1, and sodA). We established a clonal group (CG) threshold based on 104 allelic differences; a total of 56 CGs were identified from among 263 C. striatum strains. We also defined an outbreak threshold based on seven allelic differences that is capable of identifying closely related isolates that could give clues on hospital transmission. According to the results of analysis of drug-resistant genes and virulence genes, we identified CG4, CG5, CG26, CG28, and CG55 as potentially hypervirulent and multidrug-resistant CGs of C. striatum. This study provides valuable genomic epidemiological data on the diversity, resistance, and virulence profiles of this potentially pathogenic microorganism. IMPORTANCE Recently, WGS of many human and animal pathogens has been successfully used to investigate microbial outbreaks. The cgMLST schema are powerful genotyping tools that can be used to investigate potential epidemics and provide classification of the strains precise and reliable. In this study, we proposed the development of a cgMLST typing scheme for C. striatum, and then we evaluated this scheme for its applicability to hospital transmission investigations. This report describes the first cgMLST schema for C. striatum. The analysis of hospital transmission of C. striatum based on cgMLST methods has important clinical epidemiological significance for improving nosocomial infection monitoring of C. striatum and in-depth understanding of its nosocomial transmission routes.

Clinical Forms of Japanese Spotted Fever from Case-Series Study, Zigui County, Hubei Province, China, 2021.

We report a case-series study of 5 patients with Japanese spotted fever from the Three Gorges Area in China, including 1 fatal case. Seroprevalence of Rickettsia japonica was ≈21% among the local population. Our report highlights the emerging potential threat to human health of Japanese spotted fever in the area.

The size of the susceptible pool differentiates climate effects on seasonal epidemics of bacillary dysentery.

OBJECTIVES: At present, some studies have pointed out several possible climate drivers of bacillary dysentery. However, there is a complex nonlinear interaction between climate drivers and susceptible population in the spread of diseases, which makes it challenging to detect climate drivers at the size of susceptible population. METHODS: By using empirical dynamic modeling (EDM), the climate drivers of bacillary dysentery dynamic were explored in China's five temperature zones. RESULTS: We verified the availability of climate drivers and susceptible population size on bacillary dysentery, and used this information for bacillary dysentery dynamic prediction. Moreover, we found that their respective effects increased with the increase of temperature and relative humidity, and their states (temperature and relative humidity) were different when they reached their maximum effects, and the negative effect between the effect of temperature and disease incidence increased with the change of temperature zone (from temperate zone to warm temperate zone to subtropical zone) and the climate driving effect of the temperate zone (warm temperate zone) was greater than that of the colder (temperate zone) and warmer (subtropics) zones. When we viewed from single temperature zone, the climatic effect arose only when the size of the susceptible pool was large. CONCLUSIONS: These results provide empirical evidence that the climate factors on bacillary dysentery are nonlinear, complex but dependent on the size of susceptible populations and different climate scenarios.

A Shigella sonnei clone with extensive drug resistance associated with waterborne outbreaks in China.

Antimicrobial resistance of Shigella sonnei has become a global concern. Here, we report a phylogenetic group of S. sonnei with extensive drug resistance, including a combination of multidrug resistance, coresistance to ceftriaxone and azithromycin (cefRaziR), reduced susceptibility to fluoroquinolones, and even colistin resistance (colR). This distinct clone caused six waterborne shigellosis outbreaks in China from 2015 to 2020. We collect 155 outbreak isolates and 152 sporadic isolates. The cefRaziR isolates, including outbreak strains, are mainly distributed in a distinct clade located in global Lineage III. The outbreak strains form a recently derived monophyletic group that may have emerged circa 2010. The cefRaziR and colR phenotypes are attributed to the acquisition of different plasmids, particularly the IncB/O/K/Z plasmid coharboring the blaCTX-M-14, mphA, aac(3)-IId, dfrA17, aadA5, and sul1 genes and the IncI2 plasmid with an mcr-1 gene. Genetic analyses identify 92 accessory genes and 60 single-nucleotide polymorphisms associated with the cefRaziR phenotype. Surveillance of this clone is required to determine its dissemination and threat to global public health.

Serotype Distribution, Virulence Determinants and Antimicrobial Susceptibility of Streptococcus agalactiae Isolated from Young Infants

Background: Streptococcus agalactiae (Group B Streptococcus, GBS) is the most common cause of serious infections in the first 3 months of life worldwide. The pathogenicity of GBS is closely related to serotypes, surface proteins and virulence factors, and the distribution of them may vary temporally and geographically. However, data related to GBS surface proteins and virulence determinants in China are very few. The aim of this study is to investigate the genetic characteristics of clinical GBS isolates from infected infants. Methods: We recovered GBS isolates from infected infants younger than 3 months during 2017−2021 at Maternal and Child Health Hospital of Hubei Province in China. We assessed the GBS serotypes, surface proteins, virulence determinants and antibiotic resistance genes distribution, by Multilocus sequence typing (MLST) and whole-genome sequencing analysis. Results: Among 97 isolates (81 EOD and 16 LOD), 5 serotypes were detected. Serotype III was the most represented (49.5%), followed by type Ib (20.6%). The isolates belonged to 17 different sequence types (STs) that grouped into the 8 clonal complexes (CCs). The most frequently identified ST was ST17 (23.7%). The most predominant surface protein of alpha-protein-like (alp) family (one of the protein components of the GBS surface antigen, resistant to trypsin) present was Rib (41.2%), which was mainly detected in serotype III. The srr1, which encodes Srr1 protein, was identified in 54.6% of isolates. The hvgA encoding for hypervirulent GBS adhesin can be detected in all 24 serotype III GBS. Among the pilus islands genes, 50% and 58.8% of the isolates were positive for pi-1 and pi-2a genes, respectively. The presence of pi-2b was mainly associated with serotype III/CC17 strains; 56.7% of isolates carried tetM, tetO/tetL, ermB antibiotic resistant genes. Among all the virulence genes detected, the cfb-cylE-lmb-pavA pattern was the main virulence gene profile (81.4%), mainly in serotype III/CC17. Conclusions: The whole genomic sequencing data revealed the high variation in surface proteins, determining virulence and antibiotic resistance in clinical isolates from 97 GBS infected infants. These data provide insightful characteristics of genetic features of GBS. Constant epidemiological surveillance is warranted to provide information on the GBS pathogenic dynamics and antibiotic resistance profiles in the surveyed areas for improving therapeutic outcomes.

Genotypic and Phenotypic Characteristics of Moraxella catarrhalis from Patients and Healthy Asymptomatic Participants among Preschool Children.

(1) Background: M. catarrhalis can ascend into the middle ear, where it is a prevalent causative agent of otitis media in children, or enter the lower respiratory tract, where it is associated with community-acquired pneumonia (CAP). In this study, we aimed to provide an overview of the prevalence of M. catarrhalis in preschool children. (2) Methods: M. catarrhalis strains were isolated from samples. All isolates were characterized in terms of serotypes (STs), virulence genes, multilocus sequence type, and antibiotic susceptibility. (3) Results: The percentages of strains expressing lipooligosaccharides (LOSs), serotype A, B, C, or unknown were 67.61%, 15.71%, 4.28%, and 12.38%, respectively. Among the strains, 185 (88.10%) carried ompB2, 207 (98.57%) carried ompE, and 151 (71.90%) carried ompCD. The most frequently identified STs were ST449 (n = 13), ST64 (n = 11), and ST215 (n = 10). The resistance rates to the antibiotics cefuroxime, azithromycin, and erythromycin were 43.33%, 28.10%, and 39.05%, respectively. (4) Conclusions: High prevalence of some-specific ST types and high rates of antibiotic resistance indicate the necessity for an increased vigilance of resistant strains, a rational use of antibiotics in preschool children, and most importantly, the surveillance of healthy asymptomatic participants preschool children with M. catarrhalis. Our findings provide a platform for the development of novel M. catarrhalis vaccines.

Development and Validation of a Nomogram to Predict the Probability of Venous Thromboembolism in Patients with Epithelial Ovarian Cancer.

OBJECTIVE: To identify predictive factors and develop a nomogram to predict the probability of venous thromboembolism for epithelial ovarian cancer patients. Methods: Our study cohort was composed of 208 EOC patients who had received initial treatment in Sun Yat-sen Memorial Hospital from January 2016 to March 2020. Clinicopathological variables predictive of VTE were identified using univariate logistic analysis. A multivariate logistic regression model was used to select the predictive factors used for nomogram. The accuracy of nomogram was evaluated by the Concordance index (C-index), the area under the receiver-operator characteristic (ROC) curve, area under concentration-time curve (AUC) and the calibration curve. Results: Advancing age (hazard ratio [HR], 1.042; 95% confidence interval [CI], 1.000-1.085; P = .048), higher D-dimer level (HR, 1.144; 95%CI, 1.020-1.283; P = .022), lower PR immunohistochemical positive rate (HR, 0.186; 95%CI, 0.034-1.065; P = .059) and higher Ki67 immunohistochemical positive rate (HR, 4.502; 95%CI, 1.637-12.380; P = .004) were found to be independent risk factors for VTE, and were used to construct the nomogram. The C-index for VTE prediction of the nomogram was 0.75. Conclusions: We constructed and validated a nomogram able to quantify the risk of VTE for EOC patients, which can be applied in recognizing EOC patients with high risk of VTE.

[Early identification of a clustered epidemic of typhoid fever through pathogen molecular subtyping monitoring].

Objective: To identify a suspected clustered Typhoid fever by whole genome sequencing(WGS) and pulsed field gel electrophoresis (PFGE) subtyping. Methods: The nature of the epidemic was determined by combination of subtyping results of isolates and epidemiological information. Results: Five S. typhimurium isolates showed identical PFGE patterns and almost the same whole genome sequence. Epidemiological survey showed that five cases had dined in the same restaurant on the same day. Conclusion: Combined with the longest incubation period of typhoid fever, molecular subtyping of pathogenic bacteria and the field epidemiological survey, it can be preliminarily determined that the five cases have common infection sources.

Super Dominant Pathobiontic Bacteria in the Nasopharyngeal Microbiota Cause Secondary Bacterial Infection in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease responsible for many infections worldwide. Differences in respiratory microbiota may correlate with disease severity. Samples were collected from 20 severe and 51 mild COVID-19 patients. High-throughput sequencing of the 16S rRNA gene was used to analyze the bacterial community composition of the upper and lower respiratory tracts. The indices of diversity were analyzed. When one genus accounted for >50% of reads from a sample, it was defined as a super dominant pathobiontic bacterial genus (SDPG). In the upper respiratory tract, uniformity indices were significantly higher in the mild group than in the severe group (P < 0.001). In the lower respiratory tract, uniformity indices, richness indices, and the abundance-based coverage estimator were significantly higher in the mild group than in the severe group (P < 0.001). In patients with severe COVID-19, SDPGs were detected in 40.7% of upper and 63.2% of lower respiratory tract samples. In patients with mild COVID-19, only 10.8% of upper and 8.5% of lower respiratory tract samples yielded SDPGs. SDPGs were present in both upper and lower tracts in seven patients (35.0%), among which six (30.0%) patients possessed the same SDPG in the upper and lower tracts. However, no patients with mild infections had an SDPG in both tracts. Staphylococcus, Corynebacterium, and Acinetobacter were the main SDPGs. The number of SDPGs identified differed significantly between patients with mild and severe COVID-19 (P < 0.001). SDPGs in nasopharyngeal microbiota cause secondary bacterial infection in COVID-19 patients and aggravate pneumonia. IMPORTANCE The nasopharyngeal microbiota is composed of a variety of not only the true commensal bacterial species but also the two-face pathobionts, which are one a harmless commensal bacterial species and the other a highly invasive and deadly pathogen. In a previous study, we found that the diversity of nasopharyngeal microbiota was lost in severe influenza patients. We named the genus that accounted for over 50% of microbiota abundance as super dominant pathobiontic genus, which could invade to cause severe pneumonia, leading to high fatality. Similar phenomena were found here for SARS-CoV-2 infection. The diversity of nasopharyngeal microbiota was lost in severe COVID-19 infection patients. SDPGs in nasopharyngeal microbiota were frequently detected in severe COVID-19 patients. Therefore, the SDPGs in nasopharynx microbiota might invade into low respiratory and be responsible for secondary bacterial pneumonia in patients with SARS-CoV-2 infection.

Inhibitor screening using microarray identifies the high capacity of neutralizing antibodies to Spike variants in SARS-CoV-2 infection and vaccination.

Rationale: Mutations of SARS-CoV-2, which is responsible for coronavirus disease 2019 (COVID-19), could impede drug development and reduce the efficacy of COVID-19 vaccines. Here, we developed a multiplexed Spike-ACE2 Inhibitor Screening (mSAIS) assay that can measure the neutralizing effect of antibodies across numerous variants of the coronavirus's Spike (S) protein simultaneously. Methods: The SARS-CoV-2 spike variant protein microarrays were prepared by printing 72 S variants onto a chemically-modified glass slides. The neutralization potential of purified anti-S antibodies and serum from convalescent COVID-19 patients and vaccinees to S variants were assessed with the mSAIS assay. Results: We identified new S mutations that are sensitive and resistant to neutralization. Serum from both infected and vaccinated groups with a high titer of neutralizing antibodies (NAbs) displayed a broader capacity to neutralize S variants than serum with low titer NAbs. These data were validated using serum from a large vaccinated cohort (n = 104) with a tiled S peptide microarray. In addition, similar results were obtained using a SARS-CoV-2 pseudovirus neutralization assay specific for wild-type S and five prevalent S variants (D614G, B.1.1.7, B.1.351, P.1, B.1.617.2), thus demonstrating that high antibody diversity is associated with high NAb titers. Conclusions: Our results demonstrate the utility of the mSAIS platform in screening NAbs. Moreover, we show that heterogeneous antibody populations provide a more protective effect against S variants, which may help direct COVID-19 vaccine and drug development.

Phylogenetic Analysis of Serogroup O5 Vibrio cholerae that Caused Successive Cholera Outbreaks - Guangdong Province, China, 2020-2021.

Introduction: Gastroenteritis caused by non-O1/non-O139 Vibrio cholerae exhibited an increasing trend in recent years in China. Whole genome sequence (WGS) data could play an important role both in the identification of the outbreaks and in the determination of the serogroup. Here, we present the employment of WGS data in the investigation of two outbreaks caused by non-O1/non-O139 V. cholerae in Guangdong, China, 2020-2021. Methods: We obtained the whole genome sequence of 66 V. cholerae strains isolated in two outbreaks with next generation sequencing technology. We retrieved the publicly available WGS data of non-O1/non-O139 V. cholerae from public database. We used a pipeline integrated in China Pathogen Identification Net (PIN) to complete the phylogenetic analysis. Results: Two outbreaks caused by non-O1/non-O139 V. cholerae were identified using WGS data. These V. cholerae strains were determined as serogroup O5. Type 3 and 6 secretion systems were detected in these serogroup O5 strains. These serogroup O5 strains belonged to sequence type (ST) 88. Conclusions: Our analysis indicated the risk of non-O1/non-O139 V. cholerae leading to outbreaks of diarrheal diseases. The application of genomic data played an important role in the identification of the serogroup of non-O1/non-O139 V. cholerae in the lack of antiserum, which gave an example of the application of genome data in disease surveillance and public health emergency response.

Effect and attributable burden of hot extremes on bacillary dysentery in 31 Chinese provincial capital cities.

BACKGROUND: High atmospheric temperature has been associated with the occurrence of bacillary dysentery (BD). Recent studies have suggested that hot extremes may influence health outcomes, however, none have examined the association between hot extremes and BD risk, especially at the national level. OBJECTIVES: To assess the effect and attributable burden of hot extremes on BD cases and to identify populations at high risk of BD. METHODS: Daily incident BD data of 31 provincial capital cities from 2010 to 2018 were collected from the Chinese Center for Disease Control and Prevention, weather data was obtained from the fifth generation of the European Re-Analysis Dataset. Three types of hot extremes, including hot day, hot night, and hot day and night, were defined according to single or sequential occurrence of daytime hot and nighttime hot within 24 h. A two-stage analytical strategy combined with distributed lag non-linear models (DLNM) was used to evaluate city-specific associations and national pooled estimates. RESULTS: Hot extremes were significantly associated with the risk of BD on lagged 1-6 days. The overall cumulative relative risk (RR) was 1.136 [95% confidence interval (CI): 1.022, 1.263] for hot day, 1.181 (95% CI: 1.019, 1.369) for hot night, and 1.154 (95% CI: 1.038, 1.283) for hot day and night. Northern residents, females, and children younger than or equal to 14 years old were vulnerable under hot night, southern residents were vulnerable under hot day, and males were vulnerable under hot day and night. 1.854% (95% CI: 1.294%, 2.205%) of BD cases can be attributable to hot extremes, among which, hot night accounted for a large proportion. CONCLUSIONS: Hot extremes may significantly increase the incidence risk and disease burden of BD. Type-specific protective measures should be taken to reduce the risk of BD, especially in those we found to be particularly vulnerable.

Bacteremia complicated with pneumonia caused by Corynebacterium diphtheriae.

Due to the use of vaccines, infections caused by Corynebacterium diphtheriae are now rare. Here we describe a case of bacteremia complicated with pneumonia caused by C. diphtheriae in a 69-year-old male patient. The patient presented at the emergency department with a 2 days history of fever of unknown origin. Subsequent examinations revealed pneumonia and bacteremia. Non-toxigenic C. diphtheriae strains were isolated from blood and sputum. The patient had antimicrobial therapy with good improvement. We highlight the important role of C. diphtheriae in causing bacteremia and pneumonia, and its accurate and timely diagnosis is needed to avoid poor visual outcomes.

A Novel Strategy for the Detection of SARS-CoV-2 Variants Based on Multiplex PCR-Mass Spectrometry Minisequencing Technology.

The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2 variant-carrying plasmid, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q, and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants. Twenty-one respiratory tract pathogens (9 bacteria and 12 respiratory viruses) and nucleic acid samples from non-COVID-19 patients were selected for specific validation. Twenty samples from COVID-19 patients were used to verify the accuracy of this method. The 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and six variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1 (Gamma) and B.1.617.2 (Delta), could be identified. The detection limit for all 9 sites was 1.5 × 103 copies. The specificity of this method was 100%, and the accuracy of real-time PCR cycle threshold (CT) values less than 27 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge. mPCR-MS minisequencing provides a new detection option with practical application value for SARS-CoV-2 and its variant infection. IMPORTANCE The emergence of SARS-CoV-2 variants is the key factor in the second wave of the COVID-19 pandemic. An all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-mass spectrometry minisequencing system was developed in this study. Six SARS-CoV-2 variants (Alpha, Beta, Epsilon, Iota, Gamma, and Delta) can be identified simultaneously. This method can not only achieve the multisite simultaneous detection that cannot be realized by PCR coupled with first-generation sequencing technology and quantitative PCR (qPCR) technology but also avoid the shortcomings of time-consuming, high-cost, and high technical requirements of whole-genome sequencing technology. As a simple screening assay for monitoring the emergence and spread of SARS-CoV-2 and variants, mPCR-MS minisequencing is expected to play an important role in the detection and monitoring of SARS-CoV-2 infection as a supplementary technology.